Metal-catalyzed oxidation (MCO) of proteins is believed to play an important role in physiopathology and aging. Oxidative damage affects a variety of amino acid side chains and, in some instances, results in introduction of carbonyl functions. These moieties are of particular interest as their quantification by spectrophotometric methods after reaction with 2,4-dinitrophenylhydrazine has become the standard method to assess protein oxidation. One objective of the present project is the chemical characterization of protein oxidation products, with special emphasis on protein carbonyls. In previous progress reports, I described the development of analytical methods for quantitation of glutamic and aminoadipic semialdehydes, two specific carbonyl products derived from arginine and proline an from lysine, respectively. The methods are based on selected ion monitoring gas chromatography/mass spectrometry after isotopic dilution and are applied to protein hydrolysates. Analyses have been extended to additional model proteins (RNase and lysozyme) oxidized in vitro and to new tissue samples. Results confirm that glutamic and aminoadipic semialdehydes account for the majority of protein carbonyls after metal-catalyzed oxidation in vitro and that oxidized bovine serum albumin, in which they make up only about one-half of the total carbonyl value, is an exception and not the rule. Analyses of rat liver extracts have been repeated and have confirmed that aminoadipic and glutamic semialdehydes are present at similar concentrations, and that they represent also about one-half of the total carbonyl value. Analyses of additional tissue proteins (mouse liver, lung, and brain) are in progress. The second objective of this project is the characterization of oxidative modification and damage in specific proteins. Studies on recombinant Syrian hamster SHa(29-231) prion protein were initiated given the fact that copper (II) binding is the only known functional property of the prion protein, and that transition metal binding facilitates MCO. In the previous report, I described the striking susceptibility of the protein to oxidation using the standard ascorbate/Cu++ system. Subsequently, I have studied the susceptibility to oxidation of different domains of the protein using a combination of amino acid analysis, simultaneous sequencing, and proteolytic digestion coupled to liquid chromatography/mass spectrometry. Preliminary results indicating that oxidation leads to prion protein aggregation and precipitation were not confirmed, and it was determined that copper binding alone is sufficient to induce such changes under the experimental conditions used. These results indicate that metal-catalyzed oxidation of the prion protein occurs easily, and that the possible occurrence and implications of this reaction in vivo should be considered.